Flow & Imaging Cytometry Core Facility

NINDS流动和成像细胞仪核心设施（设施）由NINDS DIR于2001年建立，作为NINDS和其他内部研究人员在基础和临床研究项目中支持其工作的重要资源。该设施持续提供由该设施管理的流式细胞仪和成像设备的无缝使用，以及培训研究人员正确和最佳使用该设备的关键专业知识。设施工作人员还可以提供建设性的意见，设计和优化各种新颖的和定制的多重染色方案，使用适当的细胞表面、细胞质、细胞核、蛋白质、基因表达和生理指示染料生物标志物的独特组合，这些都是任何特定项目的初始研究者特别感兴趣的。应要求，设施工作人员定期进行多色流式和原位细胞术实验，并分析单个或多个感兴趣的细胞群的数据。应用基于不同生物标志物测试组合的用户定义布尔逻辑门控策略，该设施为每个研究者提供单个细胞或感兴趣的细胞群体中复杂信号分布的定量描述和解释。利用流式细胞仪的制备细胞分选能力，设施工作人员可根据初始研究者的要求，协助从异质细胞制剂中分离高度纯化的细胞群和亚群。这些包括从中枢神经系统和PNS组织中分选神经干细胞和祖细胞，特定的神经元和胶质细胞类型，以及非神经细胞类型（小胶质细胞，内皮细胞）。此外，该设施还常规地从骨髓、外周血、脾脏和脑脊液中分选非中枢神经系统来源的干细胞（造血、间充质和赫斯特染色-不包括侧群）和不同的白细胞表型（淋巴细胞、单核细胞、中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞、肥大细胞）。然后，这些高度纯化的细胞被NINDS和其他校内研究人员常规用于进一步的体外和体内研究，包括细胞培养、移植、延时和生理指示染料成像，以及用感兴趣的特定基因结构进行转导或转染。此外，facs纯化的细胞已被用作Western blotting和免疫沉淀实验的宝贵蛋白质来源，以及RT-PCR，微阵列，RNA- seq和microRNA测定的RNA来源。这些细胞也为使用反义和siRNA探针进行特异性功能丧失实验提供了前所未有的见解。在上述应用中使用facs纯化的均质细胞的主要意义在于，这种方法减少了在研究异质细胞制备时固有的发现的模糊性。总的来说，该设施提供的定量流动和原位细胞测定的常规应用以及制备细胞分选服务为NINDS和其他校内研究人员提供了许多细胞生物学方面的见解，这是无法通过其他方式证明的。自2001年成立以来，该基金为500多名NINDS和其他内部研究人员、研究员、学生和科学人员提供了这些宝贵的服务，并支持了来自15个NIH研究所和中心105个实验室的700多个基础、转化和临床研究项目的多学科科学研究，最终在备受赞誉的生物医学和生物科学期刊上发表了230多篇同行评议文章。在过去12个月中，该基金支持了141名研究人员、研究员、学生和实验室工作人员的研究需要，为32个研究所和其他校内实验室的133个基础科学、临床和转化研究项目作出了贡献，并促进了研究所与美国国立卫生研究院其他6个研究所和中心之间的多学科合作项目。在过去12个月里，该基金的贡献被纳入了在备受赞誉的科学期刊上发表的16篇同行评议文章中，并加速了另外18篇文章的编写，目前正在准备提交期刊或在期刊同行评议中。

NINDS Flow and Imaging Cytometry Core Facility (Facility) was established in 2001 by NINDS DIR as a crucial resource to NINDS and other intramural investigators to support their work in both basic and clinical research programs. The Facility continuously provides seamless use of flow cytometry and imaging equipment managed by the Facility as well as the crucial expertise to train investigators in proper and optimal use of this equipment. The Facility staff is also available to provide constructive input in designing and optimization of a variety of novel and customized multiplex staining protocols using unique combinations of appropriate cell surface, cytoplasmic, nuclear, protein, gene expression and physiological indicator dye biomarkers that are of specific interest to initiating investigator(s) for any given project. When requested, the Facility staff routinely carry out multi-color flow and in situ cytometry experiments and analyze the data from single or multiple cell populations of interest. Applying user-defined Boolean logic gating strategies based on different combinations of biomarkers tested, the Facility provides quantitative accounts and interpretations of the complex signal distributions in individual cells or cell populations of interest to each investigator. Using the preparative cell sorting capability of flow cytometers, the Facility staff is available to assists with isolation of highly purified cell populations and subpopulations from heterogeneous cell preparations, as requested by the initiating investigator. These include sorting of neural stem and progenitor cells, specific neuronal and glial cell types, as well as non-neural cell types (microglia, endothelial cells) from both CNS and PNS tissues. In addition, the Facility routinely sorts non-CNS-derived stem cells (hematopoietic, mesenchymal and Hoechst dye-excluding side populations) and different leukocyte phenotypes (lymphocytes, monocytes, neutrophils, basophils, eosinophils, mast cells) from the bone marrow, peripheral blood, spleen, and cerebrospinal fluid. These highly-purified cells of interest are then routinely used by NINDS and other intramural investigators for further in vitro and in vivo studies including cell culture, transplantation, time-lapse and physiological indicator dye imaging, and for transduction or transfection with specific gene constructs of interest. Additionally, FACS-purified cells have been utilized as an invaluable source of proteins for Western blotting and immunoprecipitation experiments as well as a source of RNA for RT-PCR, microarray, RNA-Seq, and microRNA assays. These cells have also provided unprecedented insights into specific loss-of-function experiments using antisense and siRNA probes. The major significance in using FACS-purified homogeneous cells in the abovementioned applications is that this approach reduces the ambiguity in the findings inherently present when studying heterogeneous cell preparations. Taken together, the routine applications of quantitative flow and in situ cytometry assays and preparative cell sorting services provided by this Facility has produced numerous insights into cell biology for NINDS and other intramural investigators, which could not otherwise be demonstrated. Since inception in 2001, the Facility has provided these invaluable services to more than 500 NINDS and other intramural investigators, fellows, students, and scientific staff and supported the multidisciplinary scientific research in over 700 basic, translational, and clinical research projects from 105 laboratories across 15 NIH institutes and centers, culminating in publication of over 230 peer-reviewed articles in highly acclaimed biomedical and biological scientific journals. Over the past 12 months, the Facility supported the research needs of 141 investigators, fellows, students, and laboratory staff contributing to 133 basic science, clinical and translational research projects in 32 NINDS and other intramural laboratories and facilitated multidisciplinary collaborative projects between NINDS and 6 other NIH institutes and centers. Contributions by this Facility over the past 12 months were incorporated in publications of 16 peer-reviewed articles in highly acclaimed scientific journals and expedited the preparation of 18 more articles currently in preparation for journal submission or in journal peer review.